Wound healing, for example of damaged skin or mucous membrane, usually proceeds in three phases: the inflammation phase, the proliferation phase and the reconstruction/remodelling phase. In the case of a fresh wound or skin injury which is to be treated, inflammatory processes, which encompass, in particular, the ingress of diverse inflammation factors (such as, for example, fibronectin) and cells of various types, such as, for example, monocytes, phagocytes, polymorphic cells and macrophages, take place within the first 24 hours, ultimately resulting in the formation of a fibrin matrix and vascular endothelial cells. Besides the said factors and cells, the wound secretion forming at the same time also contains cell detritus and a number of proteolytic enzymes as well as bacteria which have entered the wound and contain substances acting in this respect.
The proteolytic enzymes, some of which are highly active, are the reason why wound-healing-promoting, protein- or peptide-containing medicaments applied to the wound are often relatively or totally ineffective since the protein or polypeptide in question is deactivated, cleaved and degraded by said enzymes owing to its chemical and biological nature before it is able to develop an adequate pharmacological efficacy. The problem is additionally exacerbated by infection of the wound with bacteria or the ingress of cell debris. In all these processes, the respective half life of biodegradation of the medicament protein in question under the given physiological conditions plays a crucial role in the question of whether the said protein has adequate or adequately long efficacy in the case of damage to the skin, mucous membrane and the like if it is not administered systemically, such as, for example, subcutaneously or intravenously, but instead by application to the wound, i.e. topically.
For this reason, proteins and polypeptides which are particularly sensitive to enzymatic processes have hitherto not been employed successfully or not sufficiently successfully for topical application to skin wounds, even if it was attempted to introduce a physical barrier between the active compound and the wound and to maintain this for as long as possible, for example by means of corresponding porous membranes which only allow molecules smaller than 50,000 daltons to pass through. However, most proteolytic enzymes have a size larger than 100,000 daltons, which prevents them from migrating into the active-compound depot above the wound. Even with this measure, however, the effective half lifes of protein active compounds are very short since they are cleaved rapidly by proteolytic enzymes in the wound secretion forming on entry into the wound and are generally thereby rendered pharmacologically ineffective.
Another possibility for countering premature degradation of the active-compound protein is occasionally seen in the provision of slow-release formulations, in which the active compound is incorporated into a biodegradable polymer material, from which it is released in accordance with the kinetics of degradation of the polymer. However, these kinetics often do not correspond to the kinetics of degradation of the active compound after release into the wound.
Not least for this reason, pharmaceutical proteins are generally administered systemically, enabling their half life to be significantly extended and also transporting them more rapidly to the sites in the body where they are intended to develop their therapeutic efficacy. In this administration method, however, the doses of the protein-containing active compound must be sufficiently high in order to achieve the desired therapeutic effect, which often inevitably results in undesired side reactions.
In the case of therapeutic treatment of skin injuries, systemic administration of an active compound appears, in addition, less appropriate in principle since the healing effect of the medicament is actually only necessary locally. There is thus a general problem if protein-containing active compounds are to be employed for the treatment of skin injuries and open flesh and skin wounds.
Protein active compounds for use in injuries of this type, as can occur in the case of violent mechanical impacts and irritation and in burns and scalds, are known in principle. The use of growth factors, such as, for example, EGF, TGF beta, GCSF, GM-CSF, HGH, CNTF, EPO or TPO, in the healing of such conditions has thus recently been discussed.
In particular, importance is increasingly being attached to the non-haematopoietic action of erythropoietin (EPO) in connection, for example, with the stimulated formation and regeneration of endothelial and tissue cells, such as connective tissue, muscle tissue, epithelial tissue and nerve tissue, which has not been known for very long.
Thus, WO 2004/001023 describes, inter alia, the use of EPO and TPO for stimulating vascular re-formation and tissue regeneration and improving wound healing, for example after operations or injuries.
WO 2005/063965 teaches the use of EPO for the targeted structurally controlled regeneration of traumatised tissue, where topical or transdermal administration of the active compound is also proposed, where not only is endothelial cell growth stimulated, but also parenchymal regeneration and the formation of the wall structures are promoted, so that coordinated three-dimensional growth for the build-up of a functional tissue, organ or parts thereof takes place.
WO 2005/070450 describes the use of EPO in the regeneration of vessels and tissue with a weekly dose of less than 90 IU/kg of body weight for the area of wound treatment also. Although possible topical application is in principle mentioned here, it is nevertheless emphasized that systemic administration is preferred.
Haroon et al. (American J. Pathol. 2003, 163, 993) discuss the novel role of EPO in wound-healing processes induced by fibrin.
In a review article, Brines and Cerami (Kidney International, 2006) discuss the role of EPO in the protection of tissue.
Erythropoietin, EPO derivatives, such as, for example, pegylated or dimerized EPO (for example WO 02/49673 or WO 01/02017), and presumably also correspondingly active synthetic EPO peptide mimetics (as known, for example, from WO 96/40749, WO 96/40772, WO 01/38342, WO 01/091780, WO 2004/101611, WO 2004/100997, WO 2004/101600, WO 2004/101606 and WO 2006/050959) thus appear to be highly suitable for specifically initiating and controlling the re-formation and regeneration of the tissue in question in the case of damage to the skin and mucous membrane, in the case of open skin and flesh wounds and also in the case of skin irritation due to burns or scalds, and ultimately promoting and accelerating healing.
It would thus be desirable to have available EPO, EPO derivatives, EPO peptide mimetics and other protein or peptide active compounds with a similar or different action for these applications in the form of a preparation to be applied topically. Since the half life of EPO in plasma is only about 48 hours, an inadequate or at least unsatisfactory action generally arises in the case of topical use, which cannot be significantly improved even by pegylation or dimerization of the molecule and the consequent extension of the plasma half lifes.
The object is thus to provide, in particular, EPO and its bioequivalent derivatives, fragments, mimetics and the like, but also other proteins or peptides which are suitable or effective for wound healing, for topical use in the said wound indications without dramatic losses of activity occurring due to proteolysis owing to enzymatic or other processes in the wound.